HIF-1 controls the transcription of many genes that are involved in key aspects of cancer biology. Overexpression of HIF-11, an inducible subunit of HIF-1 in response to hypoxia, is a prognostic factor in many cancers. The major signaling axis mediated by PI3K, PDK1, and Akt (also referred as protein kinase B) plays a significant role in the regulation of HIF-11 expression. Both GSK32 and MAPKs are also known to directly phosphorylate HIF-11, thereby affecting its stability and/or nuclear localization. To understand the regulatory network that suppresses tumor development and tumor angiogenesis, we have recently identified HIF-11 as a new in vitro substrate of Polo-like kinase 3 (Plk3). Plk3 strongly phosphorylates HIF-11 on serines 576 and 657 in vitro, two residues that lie in the oxygen-dependent degradation domain and near the nuclear export signal, respectively. By studying primary isogenic murine embryonic fibroblasts (MEFs), we have shown that PLK3-/- MEFs are hyper-sensitive to the induction of HIF-11 under hypoxia or treated with nickel, a hypoxia mimetic. Compared with that of wild- type MEFs, PLK3-/- MEFs contain a high level of Akt1/PKB activities, which is tightly associated with the inhibitory phosphorylation of GSK32. Consistent with the potential role of Plk3 in regulating the hypoxia signaling network, PLK3-/- mice develop tumors in various organs at an advanced age and PLK3-/- tumors are large in size and highly vascularized, suggesting active tumor angiogenesis. On the basis of these observations regarding physical and functional interactions between Plk3 and HIF-11, we hypothesize that Plk3 negatively regulates the hypoxia regulatory network and HIF-11-dependent tumor angiogenesis. To test the validity of this hypothesis, we will (i) study functional interaction between Plk3 and known signaling molecules, including GSK32 and MAPKs that phosphorylate HIF-11, and identify additional Plk3 target(s) upstream of Akt1, (ii) determine whether PLK3-/- mice are prone to tumorigenesis under hypoxia, and (iii) investigate whether mice harboring Plk3 phosphorylation-resistant mutant alleles of HIF-11 are more susceptible to tumorigenesis after nickel exposure. The combined in vitro and in vivo studies will greatly facilitate the elucidation of a new mechanism by which HIF-11 is regulated by Plk3 during hypoxic responses or after exposure to environmental carcinogens such as nickel compounds. A detailed understanding of the molecular regulation of HIF-11 will add significantly to the existing knowledge of tumor angiogenesis and tumor cell resistance to anti-cancer therapies. PUBLIC HEALTH RELEVANCE: Polo-like kinases function to regulate various aspects of cell growth and cell death. Our recent biochemical and mouse genetic studies show that Plk3 plays an important role in the regulation of the HIF- 11 signaling pathway, as well as in suppressing tumorigenesis. The major goal of this project is to elucidate what signaling pathways mediate Plk3 function in hypoxic stress responses and how genotoxic metal toxicants usurp cellular mechanisms that regulate HIF-11 during hypoxia.